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Seed cryobiotechnology –
fundamental aspects
Hugh W. Pritchard
Royal Botanic Gardens Kew, Wakehurst Place, UK
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Introduction – seed types
Ger
min
atio
n (%
)
0
20
40
60
80
100
V. paradoxa
S. cuminii
D. caffra
S. cocculoides
Khaya senegalensis
Moisture content - fresh weight basis (%)
0 10 20 30 40 50
0
20
40
60
80
100
T. emetica
L. microcarpa
S. birrea
X. americana
K. africana
A
B
3-7 % MC, preferably -18°C for seed
banking
Recalcitrant
Ort
ho
do
x
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Outline
1) History 19th Century – latent life of seeds 20th Century – cryopreservation 21st Century - cryobiotechnology
2) Policy arena Aichi Biodiversity Targets
GSPC; PSESP; Exceptional Species GPA FGR ITPGRFA
2) Fundamental aspects
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1) History – 19th Century at Kew (1899)
© 2015 Royal Botanic Gardens, Kew
William Turner Thiselton-Dyer (3rd Director of Kew)
4
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1) History – 19th century at Kew (1899)
© 2015 Royal Botanic Gardens, Kew
Mustard seed 88% germination; all others 100%
Thiselton-Dyer (1899) Proc. Royal Soc., Lond. 5
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Germination “unimpaired”, but some seeds can be brittle
Poppy
Cryogen, temperature / time
Species Reference
‘froze mercury’ (-39°C) / 15 min
wheat, barley, rye, broad bean
Edwards & Colin (1834)
-57 or –110°C / 30 or 20 min
9 species, including California poppy
Wartman (1860)
liquid air / 110 h
12 sorts from wide range of families
Brown & Escombe (1897)
liquid hydrogen (-250°C) / 1 – 6 h
several kinds Thiselton-Dyer (1899)
liquid air / 176 days
sweet clover Busse (1930)
1) Late 19th and early 20th century advances
6
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3 32 406
3230
7830
13400
0
5000
10000
15000
Num
ber
of
refe
rences
to p
lant
cry
opre
se
rvation
Decade of publication
1) History - 20th century plant cryopreservation
© 2015 Royal Botanic Gardens, Kew
Google Scholar
7
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Decade No. of
papers Material
1955-1964 0 -
1965-1974 0 -
1975-1984 8 cultured plant cells = protoplasts = meristems
1985-1994 95 cultured plant cells > meristems > zygotic embryos > somatic embryos > winter buds = pollen
1995-2004 269 cultured plant cells > meristems > zygotic embryos > somatic embryos > winter buds
2005-2014 603 meristems > zygotic embryos (including seeds and axes) > somatic embryos > cultured plant cells = protocorms > winter buds = pollen;
© 2015 Royal Botanic Gardens, Kew
1) History - 20th century expansion of plant cryopreservation: Web of Science
8
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1) 21st Century - cryobiotechnology
July 2017 (Acta Horticulturae)
January 2016 (Biotech Adv)
Introduction of the term
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WANG Qiaochun
March 2018 (Biodiv & Cons)
May 2018 (Plant Cell Rep)
Gayle Volk
1) 21st Century - cryobiotechnology
Use of the term
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Statistics. Definition: Biotechnology ……as the application of science and technology to living organisms as well as parts, products and models thereof, to alter living or non-living materials for the production of knowledge, goods and services.
Biotechnology……. as the application of biological organisms, systems, or processes by various industries to learning about the science of life and the improvement of the value of materials and organisms such as pharmaceuticals, crops, and livestock.
Biotechnology… .means any technological application that uses biological systems, living organisms, or derivatives thereof, to make or modify products or processes for specific use.
1) 21st Century - cryobiotechnology
Developing the definition of the term
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Definition Cryobiotechnology……. is the use of modern technologies to understand the response of biological systems to low temperature (cryo) environments, whether natural or imposed, and leading to the production of knowledge, goods and services, including the preservation of cells and tissues for use by industry, agriculture, medical science and conservation agencies
Cryobiotechnology
Natural processes
Adaptation, resilience, FPD,
supercooling, etc.
Fundamental science
Omics, structural biology, in vitro,
model systems, etc.
Applied cryopreservation &
technology
Biobanking for agriculture, medicine,
conservation; cryotherapy; design (D-
plate, etc.)
1) 21st Century - cryobiotechnology
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2014 PLOS ONE: 90 reads (c. 20 y-1)
Reads on ResearchGate with ‘cryopreservation’ in title or keywords
2016 Plant Diversity: 289 reads (c. 100 y-1)
2016 CryoLetters: 171 reads (c. 60 y-1)
1) 21st Century - cryobiotechnology
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2017 Acta Horticulturae:
428 reads (> 200 y-1)
2016 Biotech Adv: 1255 reads (> 350 y-1)
Cryobiotechnology in title
Reads on ResearchGate with ‘cryobiotechnology’ in title
1) 21st Century - cryobiotechnology
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2) Policy – Conventional on Biological Diversity
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2) Policy - Aichi Biodiversity Targets: CBD Aichi Targets: Strategic Goal C:
Improve the status of biodiversity by safeguarding
ecosystems, species and genetic diversity
16
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2). Policy – PSESP (China)
120 sp = 70 shrubs / trees; 1 fern; 38 orchids; 11 cycads
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2). Policy - Exceptional Species
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2) Policy - GSPC and many threatened plants
Wyse et al. (2018) Nature Plants
Recalcitrant
Orthodox (-20°C)
Pro
po
rtio
n o
f sp
ecie
s
The 75% target is unattainable without
urgent investment into alternative techniques.
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2). Global Plan of Action for FGR
27 priorities
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2). International Treaty for PGR for Food and Agriculture
Annex I • 64 crops or crop complexes
• 76 genera (Agropyron, Lathyrus, Solanum)
Pritchard (2016) Ind J of PGR 29, 292-7
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2). Policy – ITPGRFA (Pritchard, 2016)
Information on c. 200 species. Notable exceptions: Canavalia ensiformis, Coronilla varia
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3). Fundamental Aspects – drying and cooling
Camellia
sinensis Litchi
chinensis
Castanea
mollissima Pisum
sativum
Eriobotrya
japonica
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3). Fundamental - Seeds are hygroscopic: lose (and take up) water
Air
Wet seed in air with low RH
Seed Water potential
gradient
Air
Air and seed at equilibrium
Seed
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• Seed oil content – more oil = lower MC
• RH – lower RH = lower eMC (equilibrium MC)
• Temperature – higher temp = lower the MC
• Adsorption OR desorption (hysteresis)
What affects the extent of drying?
What affects the drying rate?
• Proximity to eMC – closer = slower rate
• Seed size and structure – great mass = slower rate
• Depth of seeds – thicker depth = slower airflow
• Air speed/ventilation – slower airflow = slower rate
• Temperature – cooler = slower rate
3). Fundamental – drying
Seeds need to be dry enough to avoid ice formation on cooling
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3). Fundamental – Isotherms (MC vs RH)
RH (%)
Mo
istu
re C
on
ten
t (
% D
W)
M
oistu
re Co
nten
t (% FW
) barley (2 % oil)
lettuce (35% oil) Isotherms are oil
content and temperature-dependent Roberts and Ellis (1989)
Ann Bot
Colder
temperature
desorption
adsorption
Zone I II III Freezable
water
Bound water
Tigh
tly-
bo
un
d w
ater
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27
3). Unfrozen water content
Landolphia kirkii axes (Pammenter et al., 1991, Plant Phys.)
0.37 g H2O /g DW
0.28 g /g
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3). Fundamental - drying methods
Options: Ambient drying (sun/shade) / Dry room / Drying cabinet / Incubator drier / Silica gel, dried rice, charcoal
Cooled incubator:
18ºC, giving an
RH of ~15%.
Protecting seeds from direct sun to reduce risk
of heat damage
Hermetically sealed room at 10-15% RH and 10 - 25C (FAO / IPGRI Genebank Standards). Handle large volume.
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3). Fundamental - Drying with desiccants
© The Hardy Orchid Society
3:1 volume desiccant: seed, i.e. dried rice, charcoal, silica gel
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3). Fundamental - Drying with controlled RH (seeds, leaves, pollen)
http://prometheuswiki
.publish.csiro.au/
RH: 5 % = silica 13% = LiCl.H2O 23 % = KAc 33 % = MgCl2 or CaCl2
40 % = Zn(NO3)2
53 % = Mg(NO3)2
66 % = NH4NO3 75 % = NaCl 85 % = KCl 94 % = KNO3
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3). Fundamental - Measuring seed moisture
• Moisture content by weighing after forced air oven drying (103°C, 17 h; 130°C, 1-4 h) by ISTA method
• Equilibrium relative humidity (RH) using Rotronic Hygrometer.
Humidity dial
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3). Fundamental – seed ageing (deaths over time)
Time units
% V
iab
ility
Sigma,
Decreasing MC (or temperature)
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3). Fundamental - Drying and longevity (days) at 60°C
Astronium urundeuva,
Medeiros et al., 1998 SS&T
KE Cw
Species 11 % MC = 1
day lifespan
(sigma, σ)
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3). Fundamental - Pre-storage RH and lifespan
10 %
x 2
Dactylorhiza fuchsii
Dendrobium anosmum
Broken stick
3 Orchids at 40°C
Pritchard et al.
(1999) Lindleyana
2nd ‘rule’ = 10% decrease
in RH doubles longevity:
seeds, spores, pollen
-10% =
Doubling
Broken stick?
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Steiner & Ruckenbauer (1995) SSR
Hordeum vulgare and Avena sativa
81-90% germination after 110 years
Storage at 10-15°C and 3% MC
Perez-Garcia et al (2007) SS&T
37 Brassicaceae species
High germination after 38-40 years
Storage at c. -5°C and < 3% MC (over silica gel)
3). Fundamental - ultra-dry
c. 5% eRH
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Ulmus carpinifolia
At higher temperatures,
the Q10 is about 2.
Tompsett (1986) Ann Bot
-75°C
-13°C
21°C
31°C
36°C
42°C
52°C
3). Fundamental - Longevity increases with reducing temperature
11 % MC,
31°C > 10
day lifespan
(sigma, σ)
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-75°C
-13°C
21°C
31°C
36°C
42°C
52°C
3). Fundamental - Relative effect of temperature becomes less when cooler
10°C
60°C
Ulmus carpinifolia
Beneficial effect of cooling is
relatively less as temperature
falls
Tompsett (1986) Ann Bot
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8 species from 6 families; 350 survival curves
Dickie et al. (1990) Ann Bot
3) Fundamental - Relative effect of temperature is same across species
- -
Universal
temperature
constants
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Available for 19 species (17 / 76 genera = 22%) on ITPGRFA: Beta, Brassica, Cicer, Eleusine, Helianthus, Hordeum, Malus, Oryza, Pennisetum, Phaseolus, Phleum, Pisum, Sorghum, Trifolium, Triticum, Vigna, Zea.
log σ = KE - CW logm - CHt - CQt 2
Distribution of seed deaths
(lifespan)
Sensitivity to moisture level
Sensitivity to temperature
(Ellis & Roberts, 1980; Ann Bot)
3). Fundamental - The viability equation
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http://www.kew.org/data/sid
Viability equation module:
55 species
Data: storage behaviour, weight / mass,
dispersal, germination, oil and protein
content, morphology and salt tolerance
3). Fundamental - Seed Information Database
(SID)
© 2015 Royal Botanic Gardens, Kew
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3). Fundamental – temperature Quadratic not predict survival at ultra-cold temperature
- -
Alfonse de Candolle. 19th
Century
But ultracold
not dead in
minutes! © 2015 Royal Botanic Gardens, Kew
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3). Fundamental – temperature Exponential no better an approximation!
Dickie et al., (1990)
Annals of Botany
Over estimate longevity at low temperatures?
© 2015 Royal Botanic Gardens, Kew
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3). Fundamental - biophysics
30 20 10 MC (FW)
Cryopreservation
Increasing rate of ageing
After Williams and Leopold (1989) – maize embryos
Drying
stress
Ice
1
2
3
43
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3. Structural biology (seeds): summary
Cooling
(recalcitrant seeds)
Cooling/time
(oily seeds)
Fluid
(hydrated
systems)
Glass
Lipid
crystal
Ice
Glass
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3). Fundamental - Coffea Arabica (water and lipid)
Dussert et al 2001, Physiol. Plant.
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3). Fundamental - sensitive Cuphea seeds with c. 25 °C melt and cold crystallisation
Crane et al (2003); Volk et al (2006) Planta
Pow
er /
dry
mas
s
(mW
atts
/ m
g)
Temperature (°C)
Control
Cold treated*
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*germination fell by 32-74%
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3). Fundamental - Unusual behaviour associated with specific lipids: 35 Cuphea sp.
Crane et al (2003) Planta
Cuphea
carthagenensis
Species C12+C14
(%)
(C12+C14) /
(C8+C10) Tolerant of
–18°C
C. procumbens 4 0.05 Yes
C. gaumeri 11 1.5 Yes
C. wrightii 59 2 No*
C.
carthagenensis
76 8 No*
C. parsonia 78 10 No*
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3). Longer-term cold sensitivity of dry oilseeds
Carica papaya Elaeis guineensis
Coffea arabica
Azadirachta indica
Partial desiccation tolerance but rapid viability loss at -20C, due to lipid crystallisation?
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3). Fundamental - Recalcitrant seeds
2 cm
Aesculus hippocastanum – horse chestnut.
Sapindaceae
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3) Fundamental - Recalcitrant seeds die during slow drying above unfrozen water content
Nadarajan & Pritchard (2014) PLOS One 50
Variable unfrozen
water content
Laurus nobilis
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3). Fundamental - PVS2 permeation and optimisation
PVS2: 30% glycerol, 15% DMSO, 15% ethylene glycol in M&S medium + 0.4 m sucrose
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Laurus nobilis axes
Nadarajan and Pritchard (2014) PLOS One
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Summary
• Long history of seed cryo • Numerous policy instruments to
support seed banking • Fundamental aspects that inform
approaches – • Seed types • Control water (all seeds) • Control temperature (all seeds) • Control cryoprotection (Recalcitrant
seeds) • Control lipid crystallisation
(‘Intermediate’ seeds)
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Funding
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